Noncovalent interactions are very important, especially in the field of biology. Noncovalent bonding holds the two strands of the DNA double helix together (hydrogen bonds), folds polypeptides into such secondary structures as the alpha helix and the beta conformation, enables enzymes to bind to their substrate, enables antibodies to bind to their antigen, enables transcription factors to bind to each other, enables transcription factors to bind to DNA, enables proteins (e.g., some hormones) to bind to their receptor, and permits the assembly of such macromolecular machinery as ribosomes, actin filaments, microtubules and many more.
There are three principal kinds of noncovalent forces; i.e., ionic interactions, hydrophobic interactions and hydrogen bonds.
Ionic Interactions as Exemplified by Protein Interactions
At any given pH, proteins have charged groups that may participate in binding them to each other or to other types of molecules. For example, negatively charged carboxyl groups on aspartic acid (Asp) and glutamic acid (Glu) residues may be attracted by the positively charged protonated amino groups on lysine (Lys) and arginine (Arg) residues.
Ionic interactions are highly sensitive to changes in pH. As the pH drops, H+ bind to the carboxyl groups (COO—) of aspartic acid (Asp) and glutamic acid (Glu), neutralizing their negative charge, and H+ bind to the unoccupied pair of electrons on the N atom of the amino (NH2) groups of lysine (Lys) and arginine (Arg), giving them a positive charge. The result: Not only does the net charge on the molecule change (it becomes more positive), but many of the opportunities that its side chain or main chain groups have for ionic (electrostatic) interactions with other molecules and ions are altered. As the pH rises, H+ are removed from the COOH groups of Asp and Glu, giving them a negative charge (COO—), and H+ are removed from the NH3+ groups of Lys and Arg, removing their positive charge. The result: the net charge on the molecule changes (it becomes more negative) and, again, many of the opportunities its side chain or main chain groups have for electrostatic interactions with other molecules or ions are altered.
Ionic interactions are also sensitive to salt concentration. Increasing salt concentration reduces the strength of ionic binding by providing competing ions for the charged residues.
Hydrophobic Interactions as Exemplified by Protein Interactions
The side chains (R groups) of such amino acids as phenylalanine and leucine are nonpolar and, hence, interact poorly with polar molecules like water. For this reason, most of the nonpolar residues in globular proteins are directed towards the interior of the molecule, whereas such polar groups as aspartic acid and lysine are on the surface exposed to the solvent. When nonpolar residues are exposed at the surface of two different molecules, it is energetically more favorable for their two “oily” nonpolar surfaces to approach each other closely, displacing the polar water molecules between them.
The strength of hydrophobic interactions is not appreciably affected by changes in pH or in salt concentration.
Hydrogen Bonds as Exemplified by Protein Interactions
Hydrogen bonds can form whenever a strongly electronegative atom (e.g., oxygen, nitrogen) approaches a hydrogen atom, which is covalently attached to a second strongly electronegative atom.
Some common examples: between the —C═O group and the H—N— groups of separated peptide bonds in proteins (giving rise to the alpha helix and beta configuration); between —C═O groups and hydroxyl (H—O—) groups in serine and threonine residues and the SH groups of cysteine of proteins and in sugars.
It is a characteristic of noncovalent interactions that they are individually weak but collectively strong. All three forms of noncovalent interactions are individually weak (in the order of 5 kcal/mole) as compared with a covalent bond (with its 90-100 kcal/mole of bond energy). There are types of bonds with an intermediary bond energy (i.e., between 15 and 70 kcal/mole). For the present invention, such types of bonds are considered noncovalent if they are by themselves insufficient to associate two proteinaceous molecules in a certain environment. In other words, noncovalent bonds are those of which a substantial number of interactions working together are needed to hold structures together. The limited strength that these interactions do have requires that the interacting groups can approach each other closely (an angstrom or less).
Thus, a multimer comprising two or more members is, in one aspect, said to be held together by noncovalent bonds if the two or more members are linked by at least three, and preferably at least five, bonds that each have a bond energy of less than 90-100 kcal/mole. A typical cysteine disulfide bridge linking two protein chains has a bond energy of about 65 kcal/mole. However, the strength of this bond is very dependent on the reducing/oxidizing environment. Thus, for the present invention, a multimer is said to be held together by noncovalent bonds when the bonds that link the two or more member chains each have a bond energy of less than 65 kcal/mole, and typically less than 20 kcal/mole. Usually, each of the bonds has a binding energy of around 5 kcal/mole. Thus, two or more members in a multimer that are held together by noncovalent bonds have a substantial number of noncovalent interactions working together to hold the structures together and have a surface topography that enables substantial areas of the at least two interacting surfaces to approach each other closely; that is, they must fit each other.